Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems |
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Authors: | Haiyin Wang Richard W. Jones |
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Affiliation: | (1) Plant and Microbial Biology, University of California, Koshland Hall, 94720 Berkeley, CA;(2) Vegetable Laboratory, Plant Science Institute, BARC West, USDA-ARS, 20705 Beltsville, MD |
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Abstract: | Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up. |
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Keywords: | Cellulase heterologous expression Escherichia coli Saccharomyces cerevisiae |
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